Optimization of CarG crystals : a protein involved in the biosynthesis of 1-carbapen-2-em-3-carboxylic acid (Car); a beta-lactam antibiotic

University essay from SLU/Dept. of Molecular Biology (until 131231)

Abstract: Carbapenems are one of the most widely used groups of antibiotics because of their broad spectrum against both gram positive and gram negative bacteria. The biosynthesis of the simplest carbapenem, 1-carbapen-2-em-3- carboxylic acid (Car) is controlled by an operon of eight genes, CarA-H. CarG is believed to possess a function of self-resistance but the mechanism is not solved. The structure of CarG was solved a few years ago. The aim of this study was to improve the crystal quality to open up for further binding studies on CarG. The protein was purified in a 4 step process; Ni2+-column, desalting, MonoQ and gelfiltration. MonoQ was added to the previous protocol with hope to further purify the protein. Different screening protocols were used and as in earlier studies of CarG, AmSO4 was a suitable precipitant. Crystals were obtained in a concentration range between 1.3-1.8 M AmSO4 and pH 7.5 and 8, respectively. Using these conditions seeding techniques and cocrystallization with imipenem and meropenem were performed. In this study I show that by adding MonoQ chromatography to the purification protocol bigger crystals and more single crystals can be obtained. Seeding, in drops with no substrate added, resulted in more square-shaped crystals but often much thinner crystals. Results from cocrystallization and seeding with imipenem show no improvement in crystal quality. Cocrystallization with meropenem gave clear crystals but was not further improved by seeding.

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